Electronic Supplementary Material
MATERIAL AND METHODS
Description of study organism, collection and housing
Our model systems Pachycondyla villosa and P. inversa belong to the ponerine
Neotropical species complex Pachycondyla villosa (cf. Lucas et al. 2002). Co-founding is
common in this complex (Heinze et al. 2001; D’Ettorre et al. 2005); however these
founding associations differ from those of other ants. First, young queens engage in
aggressive interactions early during the founding phase and establish a rank order
regulating the division of labour: low ranking queens forage, whereas high ranking
queens care for the brood (Kolmer & Heinze, 2000). Second, cooperation among queens
does not end with the emergence of workers, as it does in most other ant species, but may
lead to stable primary polygyny (cf. Trunzer et al. 1998). In the field, the majority of P.
villosa and P. inversa founding associations are formed by two or more queens. Field
observations in P. villosa suggest that new queens may join established associations and
groups of queens may move from one nest site to another close by (D’Ettorre et al. 2005).
In these tropical species, new queens are produced year-round.
P. villosa and P. inversa founding queens were collected in November 2005 in a cocoa
plantation at Centro de Pesquisas do Cacau, CEPLAC (Itabuna, Brazil). We collected
founding associations from cocoa pods and knotholes of trees (area sampled: 300 x 600m
with consistent vegetation). Founding associations contained usually two but sometimes
more queens (up to five). The queens were put in plastic tubes with some nest material at
the collection site. In the on-site laboratory, queens were individually marked with
enamel paint, and housed in plastic boxes (8 cm diameter). The walls of the plastic boxes
were coated with fluon® to prevent the ants from escaping. Queens were fed with honey
and grasshoppers until the experiments started (usually the day after collection), and
water supply in moistened cotton was provided throughout the experiment.
Aggression index calculation
In each encounter between pairs of queens, aggression was scored as: 0 = antennal
contact (non-aggressive investigation), 1 = mandibular opening (threat), 2 = biting
(physical attack), and 3 = stinging (gaster-curling with stinging attempts). The aggression
index of each encounter was calculated as:
n
 AI  f
i
AI 
i
i 1
F
where AIi is the aggression score as defined above, fi the frequency of each act, and F the
total number of interactions (cf. Errard & Hefetz 1997; D’Ettorre & Heinze 2005).
Chemical analysis of the cuticular profile
After the behavioural experiment ended, all P. inversa queens (N = 24) were frozen at 20˚C. The heads of these queens were extracted in 200μl pentane for 10 min and these
extracts were stored at -20˚C. In the Copenhagen (Denmark) laboratory, 2μl of each
extract were injected into an Agilent 6890N gas-chromatograph (capillary column:
Agilent HP-5MS, 30m x 25μm x 0.25μm; split-splitless injector; carrying gas helium at
1ml/min). Temperature program: 70°C to 180°C at 20°C/min, 180°C to 320°C at
5°C/min, and 320°C for 5 minutes. Compounds were identified by their mass spectra
produced by an Agilent 5975 mass selective detector coupled with the gaschromatograph (70eV electron impact ionization). We consistently identified 23 peaks
(figure S1, cf. D’Ettorre et al. 2004). For each chemical profile, the proportions of these
peaks were calculated. These were our original variables and were analysed first by
principal components analysis (PCA) and then by discriminant analysis, to determine
whether predefined groups (dominant and subordinate) could be discriminated by their
chemical profiles.
Supplementary references
D'Ettorre, P., Heinze, J., Schulz, C., Francke, W. & Ayasse, M. 2004 Does she smell like a
queen? Chemoreception of a cuticular hydrocarbon signal in the ant Pachycondyla inversa. J.
Exp. Biol. 207, 1085-1091.
D'Ettorre, P. & Heinze, J. 2005 Individual recognition in ant queens. Curr. Biol. 15, 2170-2174.
D'Ettorre, P., Kellner, K., Delabie, J.H.C. & Heinze, J. 2005 Number of queens in founding
associations of the ponerine ant Pachycondyla villosa. Insectes Soc. 52, 327–332
Errard, C. & Hefetz, A. 1997 Label familiarity and discriminatory ability of ants reared in mixed
groups. Insectes Soc. 44, 189-198.
Heinze, J., Trunzer, B., Hölldobler, B. & Delabie, J.H.C. 2001 Reproductive skew and queen
relatedness in an ant with primary polygyny. Insectes Soc. 48, 149-153.
Kolmer, K. & Heinze, J. 2000 Rank orders and division of labour among unrelated cofounding
ant queens. Proc. R. Soc. Lond. B 267, 1729-1734.
Lucas, C., Fresneau, D., Kolmer, K., Heinze, J., Delabie, J. H. C. & Pho, D. B. 2002 A
multidisciplinary approach to discriminating different taxa in the species complex
Pachycondyla villosa (Formicidae). Biol. J. Linn. Soc. 75, 249–259.
Trunzer, B., Heinze, J. & Hölldobler, B. 1998 Cooperative colony founding and experimental
primary polygyny in the ponerine ant Pachycondyla villosa. Insectes Soc. 45, 267-276.